Stimulation of denervated rat soleus muscle with fast and slow activity patterns induces different expression of acetylcholinesterase molecular forms

The relative amount and distribution of acetylcholinesterase (AChE) molecular forms were studied in slow soleus and (less extensively) in fast extensor digitorum longus (EDL) muscles of the rat before and after denervation and direct stimulation. Normal EDL muscles showed higher total and specific AChE activity than normal soleus muscles and contained essentially three different molecular AChE forms (G1, G4, and A12) as opposed to six forms (G1, G2, G4, A4, A8, and A12) in the soleus. Denervation reduced AChE activity in both muscles. In the soleus direct stimulation starting 2 to 3 weeks after denervation increased the specific AChE activity markedly. The increase started 12 to 24 hr after the onset of stimulation, reached 3 to 5 times normal values after 2 to 7 days, and then declined gradually toward normal values over the next 2 weeks. Furthermore, the effect on the different molecular forms depended strongly on the stimulus pattern. Thus, intermittent 100 Hz stimulation (fast pattern) induced essentially the three forms typical of the normal EDL, whereas continuous 10 Hz stimulation induced the six forms characteristic of normal soleus muscles but with some differences in their relative proportions. In the EDL, 2 days of continuous 10 Hz stimulation (the only duration and pattern examined) failed to induce a similar increase in AChE activity.     

Effect of precocious locomotor activity on the development of motoneurones and motor units of slow and fast muscles in rat

We have investigated the effect of precociously increasing locomotor activity during early postnatal development by daily treatment with the monoaminergic precursor L-DOPA on the survival of motoneurones supplying the slow soleus (SOL) muscle and the fast, tibialis anterior (TA) and extensor digitorum longus (EDL) muscles as well as the contractile and histochemical properties of these muscles. L-DOPA treatment resulted in a significant loss of motoneurones to the slow SOL muscle, but not to the fast TA and EDL muscles. Moreover, motoneurones to fast muscles also die as when exposed to increased activity in early life, if their axons are repeatedly injured. The loss of normal soleus motoneurones was accompanied by an increase in force of the remaining motor units and sprouting of the surviving axons suggesting a remodelling of motor unit organisation. The time to peak contraction of both SOL and EDL muscles from L-DOPA treated rats was prolonged at 8 weeks of age. At 4 weeks the soleus muscles of the L-DOPA treated animal developed more tension than the saline treated one. This difference between the two groups did not persist and by 8 weeks of age the muscle weight and tetanic tension from either group were not significantly different from control animals. The present study shows that early transient, precocious locomotor activity induced by L-DOPA is damaging to normal soleus but not to normal EDL/TA motoneurones.     

Recovery of rat skeletal muscles after partial denervation is enhanced by treatment with nifedipine

Following partial denervation of adult rat skeletal muscle intact axons sprout to reinnervate denervated muscle fibres and increase their territory. The extent of this increase is limited and may depend on the ability of axon terminals to form and maintain synaptic contacts with the denervated muscle fibres. Here we tested the possibility whether reducing Ca²⁺ entry into presynaptic nerve terminals through dihydropyridine sensitive channels may allow more nerve–muscle contacts to be formed and maintained. Hindlimb muscles of adult Wistar rats were partially denervated by removing a small segment of the L4 or L5 spinal nerve on one side. A nifedipine-containing silastic rubber strip was subsequently implanted close to the partially denervated soleus or extensor digitorum longus (EDL) muscles in some animals. In control experiments silastic strips which did not contain nifedipine were used. Several weeks later isometric contractions were recorded, to determine the effect of (a) partial denervation and (b) nifedipine treatment on force output and motor unit numbers. The tension produced by nifedipine treated partially denervated muscles was 82% and 79% of the unoperated contralateral value for soleus and EDL, respectively. This was significantly greater than in untreated muscles, which only produced 61% and 48%, respectively. Mean motor unit force was also significantly larger with nifedipine treatment. Histological analysis revealed that a significantly larger proportion of the total number of muscle fibres remained in nifedipine-treated partially denervated muscles (soleus, 90% and EDL, 101%) compared with untreated muscles (soleus, 51% and EDL, 66%). Thus the number of neuromuscular contacts was increased with nifedipine treatment.     

Dynamics of nuclei of muscle fibers and connective tissue cells in normal and denervated rat muscles

The mitotic activity in muscles of growing rats and the effect of denervation were studied by means of continuous infusion of 5-bromo-2-deoxyuridine (BRDU). Denervated muscles after 10 weeks contained 20 to 60% fewer muscle nuclei than normal; BRDU labeled about 25% of the nuclei of normal soleus and extensor digitorum longus (EDL) and of denervated EDL muscles but only 5% in the denervated soleus muscle. Labeled nuclei persisted in denervated but not in normal muscles. After the main growth period, the turnover of myonuclei was at most 1 to 2% per week. The behavior of connective tissue nuclei was similar to that in muscle fibers. Infusion of BRDU had no effect on contractile properties. It is suggested that the exceptionally rapid atrophy of the denervated rat soleus associated with loss of satellite cells was due to loss of myonuclei and differentiation and fusion of satellite cells. The cause may possibly be that the phase of postdenervation fibrillation is shorter than in other muscles.     

Oxidative Enzyme Activity and Soma Size in Motoneurons Innervating the Rat Slow-Twitch and Fast-Twitch Muscles After Chronic Activity

The effects of chronic activity induced by running training on the activity of the mitochondrial enzyme succinate dehydrogenase (SDH) and soma size in motoneurons innervating the slow-twitch soleus (SOL) and fast-twitch extensor digitorum longus (EDL) muscles were studied in rats using the retrograde neuronal tracer Nuclear Yellow. Rats were assigned to control and trained groups that were subjected to treadmill running for 10 weeks (2 h/day, 30 m/min, 5 days/week). After training, both SOL and EDL muscles showed clear adaptations (citrate synthase activity in the SOL muscle, and the fast-twitch oxidative-glycolytic fiber area of the EDL muscle increased significantly after training). The SDH activity of the motoneurons innervating both SOL and EDL muscles was unchanged by training. However, SOL motoneurons of trained rats had a significantly larger soma size and a significantly higher total SDH activity (SDH activity × soma size) than those of control. Total SDH activity was calculated to examine the absolute SDH protein content of the motoneurons. On the other hand, there was no difference in both soma size and total SDH activity of EDL motoneurons between the two groups. These data demonstrate that chronic activity has a considerably stronger impact on soma size and total oxidative enzyme activity of motoneurons innervating slow-twitch rather than fast-twitch muscles.     

Fatiguability and oxidative capacity of forelimb and hind limb muscles of dystrophic mice

Fatigue indices and succinic dehydrogenase (SDH) activities were determined in the extensor digitorum longus (EDL) and soleus muscles of the hind limb and the extensor carpi radialis longus of the forelimb in control and dystrophic mice aged 4 to 26 weeks. A good correlation was found between SDH activities and fatigue indices in muscles from normal mice. In the dystrophic (dy2J) mice, however, this correlation was not present. The EDL muscles from 26-week-old dy2J mice showed a much higher resistance to fatigue than age-matched controls but this was not accompanied by a significant change in SDH. The increased fatigue resistance in dy2J EDL appeared between 8 and 12 weeks of age and was temporally correlated with the onset of fused bursts of spontaneous activity in the hind limb muscles. Nevertheless, there was no conclusive evidence for a link among this spontaneous activity, oxidative enzyme capacity, and fatigue resistance.     

IGF-I treatment improves the functional properties of fast- and slow-twitch skeletal muscles from dystrophic mice

Although insulin-like growth factor-I (IGF-I) has been proposed for use by patients suffering from muscle wasting conditions, few studies have investigated the functional properties of dystrophic skeletal muscle following IGF-I treatment. 129P1 ReJ-Lama2(dy) (129 ReJ dy/dy) dystrophic mice suffer from a deficiency in the structural protein, laminin, and exhibit severe muscle wasting and weakness. We tested the hypothesis that 4 weeks of IGF-I treatment ( approximately 2 mg/kg body mass, 50 g/h via mini-osmotic pump, subcutaneously) would increase the mass and force producing capacity of skeletal muscles from dystrophic mice. IGF-I treatment increased the mass of the extensor digitorum longus (EDL) and soleus muscles of dystrophic mice by 20 and 29%, respectively, compared with untreated dystrophic mice (administered saline-vehicle only). Absolute maximum force (P(o)) of the EDL and soleus muscle was increased by 40 and 32%, respectively, following IGF-I treatment. Specific P(o) (sP(o)) was increased by 23% in the EDL muscles of treated compared with untreated mice, but in the soleus muscle sP(o) was unchanged. IGF-I treatment increased the proportion of type IIB and type IIA fibres and decreased the proportion of type I fibres in the EDL muscles of dystrophic mice. In the soleus muscles of dystrophic mice, IGF-I treatment increased the proportion of type IIA fibres and decreased the proportion of type I fibres. Average fibre cross-sectional area was increased in the EDL and soleus muscles of treated compared with untreated mice. We conclude that IGF-I treatment ameliorates muscle wasting and improves the functional properties of skeletal muscles of dystrophic mice. The findings have important implications for the role of IGF-I in ameliorating muscle wasting associated with the muscular dystrophies.     

Choline acetyltransferase and acetylcholinesterase activities in muscles of aged mice

The activities of choline acetyltransferase (CAT) and acetylcholinesterase (AChE) were assayed in intact diaphragm, extensor digitorum longus (EDL), and soleus muscles or their homogenates of young (2-6 months) and aged (24-34 months) mice. CAT activity (per mg of protein) was significantly higher in diaphragm and soleus of old mice in comparison with the young but the age change in EDL was negligible. On the other hand, AChE activity (per mg of protein) was significantly higher in EDL of old mice but in diaphragm and soleus muscles the enzyme activity did not show any significant change statistically. The diaphragm muscle was divided into two fractions, one being neuromuscular (NM) fraction and the other the remainder of the muscle (M fraction). No appreciable change in the ratio of the enzyme activities of NM fraction to the one of M fraction was obtained between the young and aged preparations. Thus, it seems likely that there is an age-related change in CAT and AChE activities which might be affected by the degree to which muscle activity is maintained.     

Factors affecting muscle fiber transformation in cross. Reinnervated muscle

Nerves of two fast muscles [peroneus longus (PL) and extensor digitorum longus (EDL)], having different type 2 muscle fiber compositions, were used to cross-reinnervate the slow soleus muscle in the rat. Contraction characteristics, histochemical muscle fiber type compsotions and myosin heavy chain (MHC) isoform compositions were determined for the reinnervated muscles. Shortening velocity increased in soleus muscles crossreinnervated with EDL nerve [X-SOL(EDL)] but not in muscles cross-reinnervated with PL nerve [X-SOL(PL)]. Type 2A MHC isoform content was increased in X-SOL(EDL) but not in X-SOL(PL), where MHC isoform composition remained similar to normal soleus. The complement of type 1 (slow) muscle fibers was reduced and that of type 2 (fast) fibers increased in both types of X-SOL muscle, but this change was significantly greater in X-SOL(EDL); the majority of the type 2 fibers in X-SOL muscles were of type 2A. Results show that “the type 2 composition” of the reinnervating motoneuron pool is an important factor in determining the transformation of a target slow muscle after cross-reinnervation. © 1993 John Wiley & Sons, Inc.     

Metabolic specialization in fast and slow muscle fibers of the developing rat

Individual fibers of prospective fast (extensor digitorum longus; EDL) and slow (soleus) muscles of rats have been analyzed to determine the profiles of key energy-generating enzymes at successive stages of postnatal development. Mean activities of lactate dehydrogenase (LDH) and adenylokinase (AK), 2 enzymes associated with contractile function, are significantly different in the 2 fiber populations at birth; furthermore, wide variations in enzyme activities exist among the individual fibers. There is a progressive refinement of enzyme levels in the soleus into a more uniform fiber population, while the fibers in the EDL progressively diverge into 2 distinct phenotypes. Changes in EDL and soleus are punctuated by periods of rapid change, with the period between 10 and 21 d being most eventful. Generally, the maturation profiles of LDH and AK coincide with the transition from neonatal to adult fast myosins and closely reflect the timing of energy demands imposed by contractile activity patterns. In contrast, activities of the oxidative enzymes malate dehydrogenase and beta-hydroxyacyl CoA dehydrogenase are similar in both muscles at birth and steadily increase during the first 3 weeks, suggesting a progressive adaptation to the aerobic extrauterine environment. After 30 d, there are differential changes in the oxidative profiles of enzymes for fatty acid and glucose metabolism. The profiles follow dietary changes associated with weaning, which suggests a phenotypic dependence of neonatal muscle on the particular available energy substrate. All enzymes are low in all fibers of EDL and soleus at birth, indicating their modest metabolic capacity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane skeleton of innervated and denervated fast- and slow-twitch muscle

We used confocal microscopy and immunoblotting to study membrane skeletal proteins of fast-twitch (extensor digitorum longus) and slow-twitch (soleus) muscles of the adult rat. In the extensor digitorum longus (EDL), beta-spectrin concentrates in costameres, whereas dystrophin is enriched at costameres but is also present in intercostameric regions. In the soleus, beta-spectrin and dystrophin underlie much of the sarcolemma, and intercostameric regions are difficult to detect. The EDL sarcolemma reorganizes following denervation to resemble soleus sarcolemma, but denervation does not significantly affect the latter. Consistent with these observations, soleus contains similar amounts of dystrophin but more beta-spectrin than EDL. Denervation increases beta-spectrin levels only in the EDL and dystrophin levels in both muscles. Denervation does not affect beta-fodrin, a beta-spectrin homolog expressed in embryonic myofibers. Thus, neuromuscular activity controls sarcolemmal organization and the levels of beta-spectrin and dystrophin, but not postnatal downregulation of beta-fodrin. The differences in organization of the sarcolemma may underlie the differential susceptibility of fast and slow myofibers to dystrophinopathies.     

Effect of denervation on sarcolemmal proteins and glycoproteins of fast and slow mammalian skeletal muscle

We compared the protein and glycoprotein composition of a sarcolemmal membrane fraction isolated from normal and denervated rat extensor digitorum longus (EDL) and soleus muscles. Membranes from EDL and soleus muscles showed significantly different protein compositions. A relatively small number of glycoproteins, which were all minor proteins, accounted for the majority of concanavalin A (ConA) and Ricinus communis agglutinin (RCA120) binding. These glycoproteins appear to be common to EDL and soleus but bound different relative amounts of lectin in the two muscles. A large proportion of the ConA binding sites in EDL, but not soleus, were cryptic (not accessible by ConA unless the membrane structure was disrupted). Denervation had a differential effect on sarcolemma from the two muscles with EDL exhibiting large changes and soleus changing little if at all. Several major proteins changed their relative concentrations after denervation and the relative amount of RCA120 bound to the major glycoproteins also changed. The major ConA-binding glycoproteins did not change in either membrane but denervation resulted in the exposure of most of the cryptic ConA-binding sites in EDL membranes. Endogenous sialyl- and galactosyl-transferase activities in the membrane fractions significantly increased in EDL, but did not change in soleus, suggesting that the turnover of the glycoproteins is increased in EDL after denervation.     

3H]Ouabain binding in normal and dystrophic mouse skeletal muscles and the effect of age

The numbers of Na+-K+ ATPase sites in skeletal muscles of normal and dystrophic mice between 3 and 17 months of age have been estimated using [3H]ouabain binding assays. In normal mice, at all ages, slow twitch muscle, soleus (SOL), bound significantly more [3H]ouabain than fast-twitch muscle, extensor digitorum longus (EDL). [3H]Ouabain binding did not alter in either SOL or EDL from normal mice over the age range studied. The numbers of Na+-K+ ATPase sites did alter in muscles taken from dystrophic mice (C57BL/6J dy2J/dy2J). In EDL there was an increase and in SOL a decrease in [3H]ouabain binding. This may be related to a change in muscle fibre metabolism from glycolytic to oxidative or to an altered activity pattern. Increasing age resulted in a progressive reduction in [3H]ouabain binding of both SOL and EDL from dystrophic mice. Part of this reduction may be only apparent and due to an increase in connective tissue composition of dystrophic muscles. A limited study of muscles from neonate dystrophic mice indicated that abnormal [3H]ouabain binding was not present in EDL before two weeks of age.     

Choline acetyltransferase activity in muscles of old rats

The total activity of choline acetyltransferase (ChAc) in the rat extensor digitorum longus (EDL) and soleus muscles increased by 50 and 55%, respectively, between 3 and 9 months of age. In rats 28 to 29 months old, the activity of ChAc in EDL and soleus diminished to 41 and 40%, respectively, of the activity observed in 9-month-old animals. Age changes of ChAc activity in the diaphragm were not significant. The number of muscle fibers in EDL and soleus muscles of rats 28 to 29 months old decreased by 44 and 38% respectively, in comparison with younger animals. Mean muscle fiber diameters did not change between 3 and 9 months of age and decreased by 24, 35 and 9% in the EDL, soleus and diaphragm, respectively, in the 28- to 29-month-old rats. The activity of ChAc expressed in relation to one muscle fiber was about the same in the EDL and soleus muscles. It increased between 3 and 9 months and decreased between 9 and 28 to 29 months of age. The observation that ChAc activity per muscle fiber was identical in the fast EDL and slow soleus muscle suggests that the physiological differences between the two muscles are not caused by a difference in the capacity of their motor nerves to synthesize ACh. In the diaphragm the activity of ChAc per muscle fiber apparently did not diminish in old age. The decrease in the total ChAc activity in the limb muscles of old animals seems due both to a decrease in the number of nerve terminals in the muscles and to a decrease in the amount of enzyme present in individual terminals. We suggest that the maintenance of ChAc activity in the motor nerve terminals in the diaphragm of old rats is due to the continuous activity of this muscle and its motor nerves.     

Carnitine and acyltransferase in experimental neurogenic atrophies: changes with treatment

Summary Carnitine level and carnitine palmityl transferase (CPT) activity were investigated in muscles of patients with infantile and juvenile spinal muscular atrophy and polyneuropathies. A significant decrease of both carnitine and CPT was found in the infantile spinal muscular atrophy, but not in the other neurogenic muscle atrophies. These findings were compared with the experimental effect of denervation and reinnervation upon the lipid metabolism in soleus and extensor digitorum longus (EDL) of adult and newborn rats. Twenty-one days after denervation free and total carnitine decreased significantly in both EDL (P<0.001) and soleus (P<0.05) of adult animals. CPT activity was significantly decreased in the soleus 50 days after denervation (P<0.005). Long-term reinnervation restored the level of carnitine fraction and CPT activity. l-carnitine treatment for 21 days restored the level of free carnitine to normal in the soleus of denervated adult animals. Denervation in newborn rats influenced carnitine concentration in soleus and EDL to a lesser extent; the treatment with l-carnitine raised short-chain acylcarnitines in denervated muscles, while reinnervation restored carnitine level within 50 days.Presented as a preliminary report at the Fifth International Congress on Neuromuscular Diseases, Marseille, France, September 1982     

Skeletal muscle fatigue and physical endurance of young and old mice

In order to evaluate the role played by muscular and extramuscular factors in the development of fatigue in old age, the time course of fatigue in isolated skeletal muscles and spontaneous motor activity and endurance of whole animals were monitored using young (3–6 months) and old (34–36 months) CF57BL/6J mice. The isolated extensor digitorum longus (EDL) and soleus muscles from old mice had smaller (P < 0.05) mass and developed lower (P < 0.02) maximal tetanic tension at 100-Hz stimulation than the muscles of young mice. During stimulation at 30 Hz every 2.5 s, a 50% decline in original tetanic tension occurred by 109 s in young EDL and 129 s in old EDL, but by 482 s in young soleus and 1134 s (projected) in old soleus, indicating more (P < 0.05) resistance to fatigue in old than young soleus. However, the old mice showed significantly fewer (P < 0.002) spontaneous ambulatory movements than the young mice. On a treadmill with a belt speed of 10 m/min at an inclination of 0°, the old mice could only run for 22 min compared to 39 min ran by young mice (P < 0.02). They took more rest periods (P < 0.02) than the young mice. In a quantitative swimming monitor, the old mice swam for a shorter (P < 0.05) time than young mice (20.4 min compared to 28.6 min). Integrated swimming activity at 20 min was smaller (P < 0.05) in old mice than in young mice (413 g/s compared to 628 g/s). Hence increased fatigue in old age is not caused by impairment of processes within the muscles, but by impairment of central or extramuscular processes. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21: 1729–1739, 1998     

Muscle fiber type differentiation and satellite cell populations in normally grown and neonatally denervated muscles in the rat

Summary To examine the neural influence upon fiber type differentiation in developing muscles, newborn rats were subjected to sciatic nerve dissection, and the denervated extensor digitorum longus (EDL) (white) and soleus (red) muscles were examined in chronologic sequence by means of histochemistry and electron microscopy. The skeletal muscles in the newborn rats were undifferentiated (type 2C fibers seen on ATPase staining) and contained numerous myotubes. In the controls, the type 2C fibers started to differentiate at around 5 days and had almost completed type differentiation by 30 days in EDL and by 90 days in soleus muscles. On the other hand, none of the fibers in the neonatally denervated muscles developed into well differentiated type 1 and 2A fibers, but both the EDL and soleus showed longlasting type 2C and 2B populations. The satellite cells in the denervated EDL and soleus muscles decreased in number at the same rate as in the control muscles with maturation. The absence of a neural supply in the developing muscles induced a delay in muscle fiber type differentiation but did not influence the satellite cell populations in either EDL or soleus muscles.     

Changes in the cholinergic system of rat sciatic nerve and skeletal muscle following suspension-induced disuse

Muscle disuse-induced changes in the cholinergic system of sciatic nerve, slow-twitch soleus (SOL), and fast-twitch extensor digitorum longus (EDL) muscles were studied in rats. Rats with hind limbs suspended for 2 to 3 weeks showed marked elevation in the activity of choline acetyltransferase in sciatic nerve (38%), in the SOL (108%), and in the EDL (67%). Acetylcholinesterase (AChE) activity in the SOL increased 163% without changing the molecular forms pattern of 4S, 10S, 12S, and 16S. No significant (P greater than 0.05) changes in the activity and molecular forms pattern of AChE were seen in the EDL or in AChE activity of sciatic nerve. Nicotinic receptor binding of [3H]acetylcholine was increased in both muscles. When measured after 3 weeks of hind limb suspension the normal distribution of type I fibers in the SOL (87%) was reduced (to 58%) and a corresponding increase in types IIa and IIb fibers occurred. In the EDL no significant change in fiber proportion was observed. Muscle activity, such as loadbearing, appeared to have a greater controlling influence on the characteristics of the slow-twitch SOL muscle than on the fast-twitch EDL muscle.     

Changes in number and distribution of orthogonal arrays during postnatal muscle development

Quantitative freeze-fracture electron microscopy was utilized to study the changes in number and distribution of orthogonal arrays (aggregates of 6-7 nm particles) of the sarcolemmas of the fast twitch extensor digitorum longus (EDL) and slow twitch soleus muscles during the first few weeks of postnatal development in the rat. In the adult rat, orthogonal arrays are present in high densities in the fast twitch type II fibers but only in low densities in slow twitch type I fibers. In this study, the changing histochemical profiles of fiber types in the EDL and soleus muscles were also determined for the first month of postnatal development and correlated with the changing number and distribution of orthogonal arrays during the same time frame. At day 3 postpartum, EDL and soleus fibers possessed few orthogonal arrays. The developing EDL fibers rapidly acquired additional orthogonal arrays until the approximate adult number and distribution were attained at postpartum day 25. In contrast, the slow twitch soleus fibers rapidly acquired orthogonal arrays and type IIA fibers until day 35 when both were in excess of adult values. Subsequently, the number of arrays and type IIA fibers declined to normal adult ranges. We suggest that the patterns of development of orthogonal arrays and fiber types are different in the EDL and soleus because the types of innervating motor units are different in the two muscles. The EDL is innervated almost entirely by fast motor units throughout early development and maturity. The soleus, however, is initially innervated by a more heterogeneous population of motor neurons. Thus, during the period of polyneuronal innervation which occurs normally during the first weeks of postnatal development, many individual soleus fibers may possess simultaneous innervation by axons from different motor neuron types. These dual influences may be responsible for the irregular pattern of development of orthogonal arrays and type IIA fibers in developing soleus fibers. Later, as the adult pattern of monosynaptic innervation is developed, expected adult values of orthogonal arrays and fiber types are attained.